In the prior cycle of this research project, we investigated the onset and spread of synchronous activity in neural networks. When we attempted to model the data, we found, as have others, that the way the network is wired together has a profound effect on how and if the network becomes synchronously active. Very little is known about how real neural networks are wired together, and essentially nothing is known about the strategies that injured networks use to rewire themselves. Tools are now available to elucidate the wiring strategies by which neurons in real networks are connected together, as well as the strategies used to repair injured networks. We developed one such tool during the last grant cycle: the organotypic hippocampal slice culture as a model of epileptogenesis and chronic epilepsy. We will employ rapid targeted path scanning multiphoton microscopy in this preparation to image arrays of transgenic neurons expressing a dual wavelength calcium fluorophore - a novel method for analyzing network connectivity. We will use these techniques and concurrent network analyses to address the following basic questions regarding network rewiring after injury: What is the underlying rewiring strategy in hippocampal areas CA3 and CA1 - do pyramidal cells tend to connect randomly, to nearest neighbors, or to heavily connected hub cells? Do neurons maintain a constant number or weight of inputs and outputs after loss of afferent and efferent connections? Do particular wiring strategies lead to epilepsy? Are more heavily connected neurons more susceptible to ictal death, and could this underlie some forms of seizure clustering? We are excited to begin addressing these fundamental questions. This information lies at the heart of epileptogenesis, and with this information in hand we can begin to develop rational strategies to prevent or reverse epileptogenesis. PUBLIC HEALTH RELEVANCE: This research will determine the strategies that neurons use to recreate synaptic connections as the brain recovers from brain injury. The results will help us understand why epilepsy occurs after some brain injuries, and will make possible the rational development of therapies to prevent epilepsy.